GRANT=6546042;P01AG The long-term objective of our research is to understand the molecular mechanisms of the pathogenic process in transmissible spongiform encephopathies (TSE) also known as prion diseases. Although the precise nature of an infectious agent in TSEs is controversial, a prevailing model is based on the ?protein-only' hypothesis. According to this hypothesis, the key event in the pathogenic process is the conversion of the prion protein from its normal, alpha-helical form, PrP-C, to a conformationally altered, protease-resistant, beta-sheet rich form, PrP-Sc. The major goal of this project is to elucidate the mechanism of the conformational conversion(s) of the prion protein and gain insight into the structure of the abnormal, beta-sheet-rich conformer. The proposed study involves experiments with the recombinant human prion protein as well as with the abnormal PrP-Sc isoform(s) isolated from diseased brain. Recently, we have shown that, under appropriate experimental conditions, the recombinant human prion protein huPrP90-231 can be converted to an oligomeric beta-sheet-rich form with physicochemical properties similar to those of brain PrP-Sc. The first specific aim is to characterize the conformational structure of this recombinant PrP-Sc model. The structure of the protein will be probed by fluorescence spectroscopic methods (fluorescence quenching, resonance energy transfer) using a series of protein variants with genetically engineered single tryptophan residues and extrinsic fluorescent probes. The second specific aim is to determine the mechanism of amyloid formation by the disease-associated, C-truncated Y145Stop variant (residues 23-144) of the human prion protein. The recombinant protein corresponding to this variant spontaneously undergoes a self-propagating transition to amyloid fibrils, providing an attractive and experimentally accessible model for studying mechanistic aspects of the conformational conversion(s) in the prion protein. The final specific aim is to determine the effect of mutations associated with inherited prion diseases on conformational properties of authentic PrP-Sc from diseased human brain. The main technique to be used for this purpose is Fourier-transform infrared spectroscopy.